Acetylation of prostaglandin synthase by aspirin.

Abstract

When microsomes of sheep or bovine semi-nal vesicles are incubated with [acetyl-3H]aspirin (acetyl sali-cylic acid), 200 Ci/mol, we observe acetylation of a single protein, as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The protein has a molecular weight of 85,000 and correspnds to a similar acetylated protein found in the particulate fraction of aspirin-treated human platelets. The aspirin-mediated acetylation reaction proceeds with the same time course and at the same concentration as does the inhibition of prostaglandin synthase (cyclo-oxygenase) (EC 1.14.99.1;8,11, Prostaglandin biosynthesis involves the initial conversion of arachidonic acid to cyclic endoperoxide intermediates designated PGG2 and PGH2 (1, 2). These relatively unstable compounds are then metabolized by other enzymes to form stable prostaglandins, or the endoperoxide intermediates are broken down to form malonaldehyde and other products (1,3). The initial conversion is catalyzed by a microsomal enzyme (or enzymes) contained in numerous tissues, including bovine and sheep seminal vesicles and human platelets (2-4). The microsomal enzyme that metabolize's arachidonic acid has been termed prostaglandin synthase or cyclo-oxygenase (EC 1.14.99.1; 8,11,14-eicosatrienoate, hydrogendonor:oxygen oxidoreductase). While the enzyme has been solubilized by neutral detergents (5-7), it has not been purified or characterized as to molecular weight or subunit structure.The anti-inflammatory effect (8) as well as the anti-platelet effect of aspirin (9) may be mediated through inhibition of prostaglandin synthesis. Recent experiments indicate that aspirin blocks conversion of arachidonic acid to cyclic endoperoxides (3, 10). Conversely, arachidonic acid prevents the inhibitory effect of aspirin on the enzyme, and the concentration of arachidonic acid that protects the enzyme correlates with the Michaelis constant of the fatty acid as cyclooxygenase substrate (11). Therefore, Rome and Lands have postulated that aspirin inhibits cyclo-oxygenase by competing with substrate for the active site (11). The inhibition by aspirin is not strictly competitive since it irreversibly inactivates cyclo-oxygenase in vitro.The mechanism by which aspirin inhibits cyclo-oxygenase is undefined. The drug is known to acetylate proteins such as albumin, immunoglobulins, and fibrinogen (12, 13). However, these reactions proceed slowly over hours and require 1-20 mM concentrations of aspirin. Aspirin inhibits cyclooxygenase under clearly different conditions; namely, within minutes at concentrations in the micromolar range (3,14).We have recently examined the relationship between protein acetylation and the inhibition of platelet function by aspirin (15). We demonstrated that aspirin acetylates a single 85,000 molecular weight protein of the particulate fraction of human platelets. The time course and aspirin concentration dependence of the acetylation reaction correlated well with the same parameters of aspirin's effect on platelet function (16)....

Clinical evidence of dose-dependent interaction between aspirin and angiotensin-converting enzyme inhibitors

“…[15][16][17][18][19][20][21][22]26 Aspirin acetylation leads to irreversible inhibition of cyclooxygenase (PGH synthase)-which is the first enzyme of PG synthesis-converting arachidonic acid to PGH2. 27,28 This enzymatic step explains the ambivalent action of aspirin which inhibits the production of both prostacyclin (vasodilator and antithrombotic) and thromboxanes (prothrombotics, broncho-and vasoconstrictors. 29,30 In general, prostaglandins play an important endogenous vasodilatory role and counteract the enhanced peripheral vasoconstriction state in hypertension and congestive heart failure.…”

Modified-Release Dipyridamole Combined with Aspirin for Secondary Stroke Prevention

“…Aspirin alters prostaglandin metabolism and irreversibly inhibits cyclooxygenase-1, suppressing thromboxane A 2 synthesis, and thus platelet function [7,8]. It is an effective antithrombotic agent even at low doses (50-100 mg/day and possibly lower) [9].…”

Structural and Functional Basis of Cyclooxygenase Inhibition

“…43 Reaction of COX with aspirin containing a radiolabeled acetyl group leads to incorporation of radioactivity into the protein. 44,45 Arachidonic acid inhibits acetylation, suggesting that aspirin acetylates a residue in the COX active site channel. [46][47][48] Ser-530 is the only residue in COX that is acetylated by aspirin, and a S530A mutant is resistant to aspirin acetylation and timedependent inactivation.…”

“…44,45 Arachidonic acid inhibits acetylation, suggesting that aspirin acetylates a residue in the COX active site channel. [46][47][48] Ser-530 is the only residue in COX that is acetylated by aspirin, and a S530A mutant is resistant to aspirin acetylation and timedependent inactivation. 49,50 Interestingly, the K m values for arachidonate binding and IC 50 values for reversible inhibition by several COX inhibitors (aspirin, flurbiprofen) are the same for both the native (Ser-530) and mutant (Ala-530) enzymes.…”